Assays used for the detection of secreted cytokines as well as other soluble substances at the protein level include the ELISA and ELISpot assays. For example, the enzyme-linked immunospot assay (ELISpot) is widely used for the detection of T-cell specific responses. In the ELISpot assay, T-cells secreting a specific cytokine are detected by incubating the T-cells in an ELISpot assay plate on which antibodies specific for the cytokine are immobilised. Cytokine bound to the antibodies is then visualised using standard immunoassay procedures. Spots of bound cytokine localised in areas of the assay plate where cytokine production has occurred indicate the presence of activated T-cells. Each spot represents cytokine production by a single cell. Therefore, if the number of cells present in the assay is known, the ELISpot assay allows the frequency of responding cell to be ascertained by counting the number of spots formed.
The ELISpot assay has also been used for other purposes such as the detection of virus infected cells, the enumeration of cells secreting specific antibody, the detection of the cells secreting fibronectin and the study of monocytes.
The use of soluble cofactors to enhance the detection of positive cells in the ELISpot assay has been described. Immobilised cofactors have been used to stimulate T-cells and the presence of cytokines in the culture medium has been detected by ELISA-based analysis.
The ELISpot assay is ideal for analytes involving low numbers of responding or producing cells. A good example of this is the analysis of specific immune response which typically involve less than 0.1% responding cells. However, the ELISpot assay has the limitation that the number of cells in an individual sample must be restricted so that only a monolayer of cells is present in the assay well (approximately 250,000 cells/well in a 96 well plate).
The ELISA assay enables the amount of cytokine produced to be quantitated. However, the ELISA assay is not useful for analytes, such as analysis of a specific immune response, where only a few cells produce the protein being detected due to its low sensitivity.